Project Summary Activation of microglia and astrocytes (glia) plays a key role in chronic pain, including neuropathic pain, by chronic release of neuroexcitatory substances such as pro-inflammatory cytokines. This pro-inflammatory milieu surrounds neurons, helping to maintain neuronal hyperexcitability that underlies chronic pain. An optimal therapeutic strategy would be to ?reset? these chronically activated proinflammatory glia to a persistent anti-inflammatory state, thus restoring normal neuronal tone. We believe that we have identified such a therapeutic; namely, adenosine 2a receptor (A2aR) agonists. Our studies, across multiple pain models, support that a single intrathecal injection of an A2aR agonist produces a remarkably enduring reversal of neuropathic pain across weeks. This efficacy is observed under conditions of pre-existing neuroinflammation, known to upregulate the expression of A2aR. The profoundly enduring resolution of neuropathic pain is a unique and striking finding, not duplicated by any therapeutic short of gene therapy. The breadth and consistency of effect across multiple models of peripheral and central neuropathic pain predicts broad clinical utility of such agents, and supports the importance of understanding how this sustained pain reversal occurs. Discovering how A2aR agonists create multi-week resolution of pain after a single dose would provide a fundamental paradigm shift in our understanding of pain regulation and redirect strategies for development of pain therapeutics. What is clear regarding mechanism is that A2aR agonists set into motion a cascade of events resulting in the suppression of ongoing neuroinflammation via the persistent, multi-week release of the potent anti- inflammatory cytokine, interleukin-10 (IL10). This creates enduring resolution of neuropathic pain, suppression of spinal proinflammatory cytokines, and suppression of spinal glial activation. From all regards, this is an unprecedented phenomenon worthy of understanding and harnessing for improving clinical pain control in males and females and, likely, more broadly for controlling negative sequelae arising from other persistent neuroinflammatory states as well. We propose mRNA and microRNA RNA-seq and epigenetic analyses in male and female rats to identify high probability mediators and candidate signaling pathways by which A2aR agonists create their enduring pain suppressive effects. Given the uniqueness of this phenomenon, we predict that the results will yield novel mediators not currently known to the pain field. By this high risk/high reward approach, these RNA-seq and epigenetic results will define the goals to be pursued in subsequent grant projects by discovering unique targets and pathways. This is a hypothesis-generating proposal seeking to understand a completely novel finding. We predict that A2aR agonism alters the transcriptional program of glial cells by creating lasting epigenetic modifications.